1. Field of the Invention
The present invention relates to fields of molecular biology and pathology. More particularly, the present invention relates to peptides of CaV2.2 and methods for their use in the treatment of pain.
2. Description of Related Art
There are six distinguishable types of voltage-dependent calcium channels (VDCC) presently described, designated L-type, N-type, P/Q-type, L-type, R-type, and T-type, which are expressed throughout the nervous system (Tsien et al., 1991). Presynaptic voltage-gated Ca2+ channels mediate rapid Ca2+ influx into the synaptic terminal that triggers synaptic vesicle exocytosis and neurotransmitter release (Llinas et al., 1981). N-type Ca2+ channels, encoded by CaV2.2 pore-forming subunit (Williams et al., 1992; Ertel et al., 2000) and P/Q-type Ca2+ channels, encoded by the CaV2.1 pore-forming subunit (Mori et al., 1991; Ertel et al., 2000), play a predominant role in supporting chemical neurotransmission in central synapses (Takahashi and Momiyama, 1993; Wheeler et al., 1994; Dunlap et al., 1995; Reuter, 1995). Sensation of pain is mediated by nociceptive neurons in the dorsal root ganglia (DRG) (McCleskey and Gold, 1999; Yaksh, 1999). N-type voltage-gated Ca2+ channels (Cav2.2) are abundantly expressed in DRG neurons (Kerr et al., 1988; Gohil et al., 1994; Westenbroek et al., 1998) and play a predominant role in control of glutamate release from DRG neurons in the spinal cord. Thus, inhibition of N-type Ca2+ channels is expected to have anti-nociceptive effect. Indeed, studies have suggested that N-type calcium channel antagonists are mainly effective in reducing pain associated with inflammation and tissue/nerve injury, although some effect has been shown in acute models of pain. Anti-nociceptive effects of L- and P/Q-type VDCC antagonists have also been reported; however, these effects appear to be moderate at best.
Pain can be essentially divided into 2 broad categories: physiological pain and pathological pain. Physiological pain is good for the organism in that it is protective. To prevent damage to tissue, physiological pain pathways are activated by noxious stimulation. Physiological pain must only be controlled under specific clinical situations, such as during surgery, medical procedures, or following trauma. Drugs that chronically disable pathways that transmit physiological pain are undesirable as they cause the organism to lose the protective function of pain. Pathological pain, on the other hand, is not the result of a noxious stimulation or healing tissue. Pathological pain originates from abnormal function of the nervous system due to nerve lesion or compression, neuropathy, tumor growth, or tissue inflammation. Current therapeutics that are used for the treatment of pathological pain are typically limited by serious side effects and the development of tolerance.
Pain researchers developed three classes of pain animal models: acute (physiological) pain model (hot plate, tail flick, paw pressure), inflammatory models (carrageenan and formalin), and nerve injury (sciatic nerve ligation, focal spinal injury) (Yaksh, 1999). A biphasic behavioural response is observed in the formalin model. The phase I of the response (1-10 min after injection) corresponds to acute afferent input resulting from the activation of primary afferent neurons. The phase II of the response (10-60 min) results from sensitization of spinal responses and considered to be an appropriate model for persistent pain (Yaksh, 1999).
Consistent with the role of N-type Ca2+ channels in pain pathway, pharmacological block of N-type Ca2+ channels by single injection or continuos infusion of synthetic ω-conopetide SNX-111 inhibited phase II formalin response in rat animal model (Malmberg and Yaksh, 1994, 1995). The role of N-type Ca2+ channels in pain pathway was further supported by analysis of Cav2.2 knockout mice (Hatakeyama et al., 2001; Kim et al., 2001; Saegusa et al., 2001). All 3 groups observed supression of phase II formalin response in Cav2.2−/− mice when compared to wild type mice.
These results pointed to N-type Ca2+ channels as potential drug target for a treatment of persistent pain. Based on this idea, Elan Pharmaceuticals (initially Neurex) developed a drug Ziconotide (SNX-111, a synthetic version of ω-conotoxin MVIIA). Very promising results were obtained with Ziconitide in clinical trails and currently FDA is considering Ziconotide for approval. However, although Ziconotide is highly effective for treatment of chronic pain, there is also a number of problems associated with its use. Ziconitide (SNX-111) is a polypeptide with a complex chemical structure and very difficult to synthesize. Ziconitide does not pass blood-brain-barrier and has to be delivered by pump infusion directly into a spinal cord, greatly limiting score of its applications. In addition, a number of severe side-effects were reported in some patients in response to Ziconitide (Penn and Paice, 2000).
It has been demonstrated that neuron-to-neuron contact is required for N-type Ca2+ channel clustering during synapse formation in rat hippocampal neuronal culture (Bahls et al., 1998). More recently, synaptic targeting of an auxiliary P/Q-type Ca2+ channel subunitβ4 was investigated (Wittemann et al., 2000). The present inventors have previously investigated targeting of recombinant N-type Ca2+ channels to synaptic locations in rat hippocampal neuronal cultures. It was found that in immature and in mature low-density hippocampal cultures, recombinant N-type Ca2+ channels are uniformly distributed in both axonal and somatodendritic compartments. In contrast, in mature high-density cultures, the recombinant N-type Ca2+ channels are clustered in presynaptic sites and primarily excluded from the somatodendritic domain. Synaptic clustering of recombinant N-type channels depended critically on the most C-terminal region of the “long” splice variant of the N-type Ca2+ channel pore-forming subunit CaV2.2a (Williams et al., 1992; Ertel et al., 2000).
In another earlier study, the inventors identified postsynaptic density-95 (PSD-95)/discs large/zona occludens-1 (PDZ) and Src homology 3 (SH3) domainbinding motifs in the same region of the CaV2.2 subunit (Maximov et al., 1999). The association of CaV2.2-NC1 C termini with the Mint1/CASK/veli-neurexin/neuroligin complex (Maximov et al., 1999) provides a possible molecular mechanism for N-type Ca2+ channel synaptic targeting during synaptogenesis, and the association of CaV2.2a-NC1 C terminal with Mint1-PDZ1 and CASK-SH3 domains (Maximov et al., 1999) links synaptic N-type channels to neurexin-neuroligin neuronal adhesion complex (Irie et al., 1997; Nguyen and Sudhof, 1997; Butz et al., 1998; Song et al., 1999) and synaptic clustering of the channels and synaptic organization (Fanning and Anderson, 1996; Kornau et al., 1997; Craven and Bredt, 1998). The importance of N-type channel association with Mint1 and neurexins is consistent with impaired presynaptic function in neurons from Mint1 kockout (Ho et al., 2003) and α-neurexins (Missler et al., 2003) knockout mice.
More recently, the inventors have shown that CaV2.2 C termini also bind to INADL-5, PAR6, and MUPP1-9 PDZ domains (Bezprozvanny and Maximov, 2001) and that the proline-rich region of the CaV2.2 C-terminus has been implicated recently in interactions with the SH3 domain of RBP (Hibino et al., 2002). Subsequently, the inventors also demonstrated that these motifs act as synergistic synaptic targeting signals for N-type channels in rat hippocampal neurons (Maximov and Bexprozvanny, 2002). The inventors also demonstrated that introduction of CaV2.2 carboxy-terminal sequence into hippocampal neurons by transfection impairs their presynaptic function (Maximov & Bezprozvanny, 2002). However, there have yet to be reported attempts to specifically block these interactions and determine the ensuing biological consequences, particular with regard to pain.